Apoptosis is the highly regulated process of programmed cell death that is essential for normal tissue development and homeostasis. It occurs in animals, humans and plants. In humans, for example, de-regulation of apoptotic processes contributes to a wide range of diseases, including disorders caused by loss of cells (e.g., neurodegenerative diseases, tissue damage from stroke or heart failure, and AIDS). Reed, J. C. and Tomaselli, K. J. (2000) Curr. Opin. Biotech., 11, 586-592. In plants, the formation of water-conducting xylem vessels, a developmental process termed xylogenesis, entails programmed cell death that appears to be a plant counterpart of animal apoptosis. Specifically, apoptosis during xylogenesis features fragmentation of nuclear DNA, recruitment of cysteine proteases and the formation of apoptotic bodies, which are distinctive and characteristic features of animal apoptosis. Roberts, K. and McCann, M. C. (2000) Current Opinion in Plant Biology, 3, 517-522; Mittler R. and Lam E. (1995) Plant Physiol, 108, 489-493; Ye. Z.-H and Varner, J. E. (1996) Plant Mol Biol., 30, 1233-1246; Minami, A. and Fukuda, H. (1995) Plant Cell Physiol, 36, 1599-1606; Beers, E. P. and Freeman, T. B. (1997) Plant Physiol., 113, 873-880; Iliev, I and Savidge R. (11999) Phytochemistry, 50, 953-960; Yu, F.-X. and Chye, M.-L. (1999) Plant J., 17, 321-327; Groover, A. and Jones, A. M. (1999) Plant Physiol., 119, 375-384; Groover, A., DeWitt, N. G., Heidel, A. and Jones, A. M. (1997) Protoplasma 196, 197-211. Inhibitors and elicitors of apoptosis have been cloned from a variety of species, but much of the apoptotic signaling pathway remains to be elucidated.
Cells undergoing apoptosis identified by morphological changes which include cell shrinkage, condensation of chromatin, nuclear fragmentation, and the formation of apoptotic bodies (Reed and Tomaselli, 2000). One of the hallmarks of apoptotic cell death is the appearance of DNA fragmentation in multiples of 200 base pairs, which results from the activation of an endonuclease that cleaves the DNA between nucleosomes. Caspases, a family of proteases, are the main executors of the apoptotic program. Two major pathways to caspase induction include activation by molecules released from mitochondria, such as cytochrome c, and activation of death receptors by extracellular signals. Newmeyer, D. D., Farschon, D. M. and Reed, J. C. (1994) Cell, 79, 353-364: O'Connor, I. Huang, D.C., O'Reilly, L. A. and Strasser, A. (2000) Curr. Opin. Cell Biol., 24, 49-52.
The unique amino acid, hypusine, is found in all examined eukaryotes and archaebacteria, but not in eubacteria, and eukaryotic initiation factor 5A (eIF-5A) is the only known hypusine-containing protein. Park, M. H. (1988) J. Biol. Chem., 263, 7447-7449; Schumann. H. and Klink, F. (1989) System Appl. Microbiol., 11, 103-107; Bartig, D., Scümann, H., and Klink, F. (1990) System. Appl. Microbiol., 13, 112-116; Gordon, E. D., Mora, R., Meredith, S.C., Lee, C. and Lindquist, S. L. (1987a) J. Biol. Chem. 262, 16585-16589. Active eIF-5A is formed in two post-translational steps: the first step is the formation of a deoxyhypusine residue by the transfer of the 4-aminobutyl moiety of spermidine to the c-amino group of a specific lysine of the precursor eIF-5A catalyzed by deoxyhypusine synthase; the second step involves the hydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylase to form hypusine.
The amino acid sequence of eIF-5A is well conserved between species, and there is strict conservation of the amino acid sequence surrounding the hypusine residue in eIF-5A which suggests that this modification may be important for survival. Park, M. H., Wolff, E. C., and J. E. Folk (1993) Biofactors, 4, 95-104. This assumption is further supported by the observation that inactivation of both of the eIF-5A's found to date in yeast, or inactivation of the deoxyhypusine synthase gene which catalyzes the first step in their activation, blocks cell division. Schnier, J. Schwelberger, H., Smit-McBride, Z., Kang, H. A., and Hershey, J. W. B. (1991) Mol. Cell. Biol., 11, 3105-3114; Sasaki, K, Abid, M. R. and Miyazaki, M. (1996) FEBS Lett., 384, 151-154; Park, M. H., Joe, Y. A. and Kang, K. R. (1998) J. Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein in yeast resulted in only a small decrease in total protein synthesis suggesting that eIF-5A may be required for the translation of specific subsets of mRNA's rather than for protein global synthesis. Kang, H. A., Schwelberger, H. G., and Hershey, J. W. B. (1993) Effect of initiation factor eIF-5A depletion on cell proliferation and protein synthesis, in Tuite, M. (ed.), Protein Synthesis and Targeting in Yeast, NATO Series H. This view is supported by the recent finding that ligands which bind eIF-5A share highly conserved motifs. Xu, A. and Chen, K. Y. (2001) J. Biol. Chem., 276, 2555-2561. The hypusine residue of modified eIF-5A was found to be essential for sequence-specific binding to RNA, and binding did not provide protection from ribonucleases. In addition, intracellular depletion of eIF-5A resulted in a significant accumulation of specific mRNAs in the nucleus, indicating that eIF-5A may be responsible for shuttling specific classes of mRNAs from the nucleus to the cytoplasm. Liu, Y. and Tartakoff, A. (1997) Supplement to Molecular Biology of the Cell, 8, 426a. Abstract no. 2476, 37th American Society for Cell Biology Annual Meeting. The accumulation of eIF-5A at nuclear pore-associated intranuclear filaments and its interaction with a general nuclear export receptor further suggest that eIF-5A is a nucleocytoplasmic shuttle protein rather than a component of polysomes. Rosorius, O., Reichart, B., Kratzer, F., Heger, P., Dabauvalle, M.-C. and Hauber, J. (1999) J. Cell Science, 112, 2369-2380.
The first cDNA for eIF-5A was cloned from human in 1989 by Smit-McBride et al., and since then cDNA's or genes for eIF-5A have been cloned from various eukaryotes including yeast, rat, chick embryo, alfalfa, and tomato. Smit-McBride, Z., Dever, T. E., Hershey, J. W. B. and Merrick, W. C. (1989a) J. Biol. Chem., 264, 1578-1583; Schnier et al. (1991) (yeast); Sano, A. (1995) in Imahori, M. et al. (eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, The Netherlands, 81-88 (rat); Rinaudo, M. S., and Park, M. H. (1992) FASEB J, 6, A453 (chick embryo); Pay, M. H., Wolff, E. C., Smit-McBride, Z., Hershey, J. W. B., and Folk, J. E. (1991) Plant Mol. Biol., 17, 927-929 (alfalfa); Wang, T. W., Lu, L., Wang, D., and Thompson J. E. (2001) J. Biol. Chem., 276, 17541-17549 (tomato).
Expression of eIF-5A mRNA has been explored in various human tissues and mammalian cell lines. For example, changes in eIF-5A expression have been observed in human fibroblast cells after addition of serum following serum deprivation. Pang, J. H. and Chen, K. Y. (1994) J. Cell Physiol., 160, 531-538. Age-related decreases in deoxyhypusine synthase activity and abundance of precursor eIF-5A have also been observed in senescing fibroblast cells, although the possibility that this reflects averaging of differential changes in isoforms was not determined. Chen, Z. P. and Chen, K. Y. (1997b) J. Cell Physiol., 170, 248-254.
Studies have shown that eIF-5A may be the cellular target of viral proteins such as the human immunodeficiency virus type 1 Rev protein and human T cell leukemia virus type 1 Rex protein. Ruhl, M., Himmelspach, M., Bahr, G. M., Himmerschmid, F., Jaksche, H., Wolff, B., Aschauer, H., Farrington, G. K., Probst, H., Bevec, D. and Hauber, J. (1993) J. Cell Biol., 123, 1309-1320; Katahira, J., Ishizaki, T., Sakai, H., Adachi, A., Yamamoto, K. and Shida, H. (1995) J. Virol, 69, 3125-3133. Preliminary studies indicate that eIF-5A may interact both with other RNA-binding proteins such as Rev, and also with Rev target RNA, suggesting that these viral proteins may recruit eIF-5A for viral RNA processing. Liu, Y. P., Nemeroff, M., Yan, Y. P. and Chen, K. Y. (1997) Biol. Signals, 6, 166-174.
Spermidine, analogs have been successfully used to inhibit deoxyhypusine synthase in vitro, as well as to inhibit the formation of hypusine in vivo, which is accompanied by an inhibition of protein synthesis and cell growth. Jakus, J., Wolff, E. C., Park, M. H., and Folk, J. E. (1993) J. Biol. Chem., 268, 13151-13159; Park, M. H., Wolff, E. C., Lee, Y. B., and Folk, J. E. (1994) J. Biol. Chem., 269, 27827-27832. Polyamines themselves, in particular putrescine and spermidine, also appear to play important roles in cellular proliferation and differentiation. Tabor, C. W. and Tabor, H. (1984) Annu. Rev. Biochem., 53, 749-790; Pegg, A. E. (1988) Cancer Res., 48, 759-774. For example, yeast mutants in which the polyamine biosynthesis pathway has been blocked are unable to grow unless provided with exogenous polyamines. Cohn, M. S., Tabor, C. W., and Tabor, H. (1980) J. Bacteriol., 134, 208-213.
Polyamines have also been shown to protect cells from the induction of apoptosis. For example, apoptosis of thymocytes has been blocked by exposure to spermidine and spermine, the mechanism of which appears to be the prevention of endonuclease activation. Desiderio, M. A., Grassilli. E., Bellesia, E., Salomoni, P. and Franceschi, C. (1995) Cell Growth Differ., 6, 505-513; Brune, B., Hartzell, P., Nicotcra, P. and Orrenius, S. (1991) Exp. Cell Res., 195, 323-329. In addition, exogenous polyamines have been shown to repress B cell receptor-mediated apoptosis as well as apoptosis in the unicellular parasite, Trypanosoma cruzi. Nitta, T., Igarashi, K., Yamashita, A., Yamamoto, M., and Yamamoto, N., (2001) Exptl. Cell Res., 265, 174-183; Piacenza, L., Peluffo, G. and Radi, R. (2001) Proc. Natl. Acad. Sci., USA, 98, 7301-7306. Low concentrations of spermine and spermidine have also been observed to reduce the number of nerve cells lost during normal development of newborn rats as well as protect the brain from neuronal damage during cerebral ischaemia. Gilad, G. M., Dornay, M. and Gilad, V. H. (1985) Brain Res., 348, 363-366; Gilad, G. M. and Gilad, V. H. (1991) Exp. Neurol., 111, 349-355. Polyamines also inhibit senescence, a form of programmed cell death, of plant tissues. Spermidine and putrescine have been shown to delay post-harvest senescence of carnation flowers and detached radish leaves. Wang, C. Y. and Baker, J. E. (1980) HortScience, 15, 805-806 (carnation flowers); Altman, A. (1982) Physiol. Plant., 54, 189-193 (detached radish leaves).
In other studies, however, induction of apoptosis has been observed in response to exogenous polyamines. For example, human breast cancer cell lines responded to a polyamine analogue by inducing apoptosis, and excess putrescine has been shown to induce apoptosis in DH23A cells. McCloskey, D. E., Casero, R. A., Jr., Woster, P. M. and Davidson, N. E. (1995) Cancer Res., 55, 3233-3236; Tome, M. E., Fiser, S. M., Payne, C. M. and E. W. Gerner (1997) Biochem. J, 328, 847-854.
Apoptosis is currently a field of intense study, and many techniques which may be useful in the control of the apoptotic process are being explored. A great deal of this research has been focused on inducing apoptosis in cancer models, but the prevention of unwanted apoptosis in disease processes is also under investigation. Activation of Jun kinase has been shown to be protective against nitric oxide-induced apoptosis in cardiac myocyte cells. Andreka, P., Zang, J., Dougherty, C., Slepak, T. I., Webster, K. A. and Bishopric, N. H. (2001) Circul. Res., 88, 305-312. Overexpression of anti-apoptotic proteins such as Bcl-2 in T and B lymphocytes has been shown to reduce apoptosis in lymphoid organs following cecal ligation and puncture, and decreased liver apoptosis has been observed in Bcl-2-overexpressing hepatocytes. Hotchkiss, R. S., Swanson, P. E., Knudson, C. M., Chang, K. C., Cobb, J. P., Osborne, D. F., Zollner, K. M., Buchman, T. G., Korsmeyer, S. J. and Karl, I. E. (1999) J. Immunol., 162, 4148-4156; Rodriguez, I., Matsuura, K., Khatib, K., Reed, J. C., Nagata, S. and Vassalli, P. (1996a) J. Exp. Med., 183, 1031-1036. Inhibitors of caspases have also been used to block Fas-mediated apoptosis in the liver. Rodriguez, I., Matsuura, K., Ody, C., Nagata, S. and Vassalli, P. (1996b) J. Exp. Med., 184, 2067-2072.
Inhibition of caspase 1 has also been observed to slow the progression neurodegenerative disease in a mouse model of Huntington's disease. Ona, V. O., Li, M., Vonsattel, J. P., Andrews, L. I., Khan, S. Q., Chung, W. M., Frey, A. S., Menon, A. S., Li, X. J. and Stieg, P. E. et al. (1999) Nature, 399, 263-267. Animal models have also been used to demonstrate the efficacy of peptidyl caspase inhibitors in stroke, myocardial infarction, sepsis, and amylotrophic lateral sclerosis. Endres, M., Namura, S., Shimizu-Sasamata, M., Waeber, C., Zhang. L., Gomez-Isla, T., Hyman, B. T. and Moskowitz, M. A. (1998) J. Cereb. Blood Flow Metab., 18, 238-247 (stroke); Wiessner, C., Sauer, D., Alaimo, D. and Allegrini, P. R. (2000) Cell. Mol. Biol., 46, 53-62 (stroke: caspase inhibitor z-VAD-tmk decreased cortical infarct by 45% after permanent middle cerebral artery occlusion in rat); Rabufetti, M., Sciorati, C., Taroxxo. G., Clementi, E., Manfredi, A. A. and Beltramo, M. (2000) J. Neurosci., 20, 4398-4404 (stroke: caspase inhibitor Ac-YVAD-cmk reduced infarct volume and decreased apoptosis, as measured by nucleosome quantitation, by approximately 50% after permanent middle cerebral artery occulusion in rats); Holly, T. A., Drincic, A., Byun, Y., Nakamura, S., Harris, K., Klocke, F. J. and Cryns, V. L. (1999) J. Mol. Cell. Cardiol., 31, 1709-1715 (myocardial infarction: systemic administration of caspase inhibitor YVAD-cmk reduced myocardial infarct size by 31% and reduced the number of apoptotic cells by approximately 70% in rabbits following coronary artery occlusion and reperfusion); Jaeschke, H., Fisher, M. A., Lawson, J. A., Simmons, C. A., Farhood, A. and Jones, D. A. (1998) J. Immunol., 160, 3480-3486 (sepsis: caspase inhibitor z-VAD attenuated apoptosis by 81 to 88% and prevented liver cell necrosis in mice experiencing endotoxin-induced liver damage); Li, M., Ona, V. O., Guegan, C., Chen, M., Jackson-Lewis, V., Andrews, L. J., Olszewski, A. J., Stieg, P. E., Lee, J. P., Predborski, S. and Friedlander, R. M. (2000) Science, 288, 335-339 (amylotrophic lateral sclerosis). These studies indicate that apoptosis can be controlled to some degree by targeting the core components of the cell-death machinery. Reed, J. C. and Tomaselli, K. J. (2000).
However, methods for controlling apoptosis and for treating disease by controlling apoptosis are still a largely unexplored area. Failures in the precise regulation of apoptosis are believed to cause or exacerbate such diverse diseases and disorders as neurological neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, Huntington's. Amyotrophic Lateral Sclerosis (Lou Gehrig's), stroke, autoimmune disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis), Duchenne Muscular Dystrophy (DMD), motor neuron disorders, ischemia, chronic heart failure, infantile spinal muscular atrophy, cardiac arrest, renal failure, atopic dermatitis, sepsis and septic shock, AIDS, hepatitis, glaucoma, diabetes (type I and type 2), asthma, retinitis pigmentosa, osteoporosis, xenograft rejection, and burn injury. Thus, methods of controlling apoptosis are needed.